Setbp1 knock-out#2
ATAC-seq library preparation was performed on FACS-sorted Lin-Kit+Sca1+ BM cells (100,000 cells per sample) using ATAC-Seq Kit (#13150, Active Motif). In short, nuclei were isolated by adding 100 L ice cold ATAClysis buffer to the cell pellet and resuspending cells with a pipette. After centrifugation (500 g, 10 min at 4C), cells were washed and incubated with the tagmentation master mix in a shaking heat block at 37C /800 rpm for 30 min. Obtained DNA was taken up in DNA purification buffer, purified using the contained DNA purification columns, amplified for 10 cycles using indexed primers, and size selected using SPRI bead solution. A quality control (QC) was performed in order to verify the size distribution of the PCR enriched library fragments. To this end, aliquots of the DNA libraries were analyzed on an Agilent Technologies 2100-Bioanalyzer, using a High Sensitivity DNA chip. This Bioanalyzer based QC was for qualitative purposes only. A precisely quantification of the libraries was done using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). The barcoded amplicons were sequenced in a NovaSeq 6000 platform (Illumina) at a depth of ~ 16M 2x150 bp reads per sample. The adapter sequences were removed from the ATAC-seq FASTQs using NGmerge (John et al., 2018). The cleaned FASTQs were aligned using Bowtie2 (2.4.4) to the mouse reference genome mm10 with the parameters very-sensitive and the duplicated reads were removed using picard (2.26.2) MarkDuplicates with REMOVE_DUPLICATES=true and Samtools. Peaks were called using Genrich with multiple steps of alignment filtering. Peak was annotated by ChIPseeker library from R/Bioconductor. Motif analysis on peak regions was performed by HOMER. DeepTools was used to generate heatmaps, profile plots, and covert bam files to bigwig files that can be visualized in IGV.